免疫荧光的方法依据你使用的试剂盒各有不同，最好你仔细阅读试剂盒的说明书按照所给程序进行操作。% P: }5 A! ?) P) ^) S/ Y
给你一个方法参考一下，不过是写的荧光双标，与单标的过程基本是类似的只是双标多标了一次。 0 A5 P! N4 E$ ^% K6 }9 b4 m4 YDouble immunofluorescence staining for BCL-6 and else. , Y# i9 {% e& A( O! lAny type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved. 1 W5 h% x/ R% O4 _" b3 I' CUse cytocentrifuged cells or frozen or paraffin dewaxed sections. , t: L4 H" _/ H. U1 Q: S; u: XPresence of endogenous fluorescent substances may affect your staining. 4 H$ U9 O( z8 s ; [: w" Q" q4 L/ H' f' Q' q6 [Note: This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP / APAAP technique. 2 F5 F; `$ M) W+ n3 g
Start at this section for unfixed or acetone-fixed specimens. # q( Z8 q' I. D2 S. S+ @
, K/ A w) E! ?! N& m4 [ o5 E& L1- Mark the slide in order to recognize the area to be stained and label the slide. , ~- k. n* l: a: p" L
2- Fix in acetone for 10 min at RT [this tep can be omitted]. + w- g7 ^: `% q8 k3- Blot the slides and let them dry for 10 min - 2 hrs. / G: R: z8 ?$ X" N: O4- Fix in 10% formalin for 10 min in a Coplin Jar. - w3 o* n g6 k8 [; P
From this point on, don't let dry the slides at any time. + d# ?3 @3 S% @5- Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK). 1 T& B4 h1 l% E# X6 n( ^
; G a& A& l, N Start at this section for dewaxed, antigen retrieved slides * `: G/ Z6 x( }7 i1 ^( m( i T9 r& j5 c6 Z; @. T
6- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes / wash. * Z Q4 A/ t9 l
7- Briefly blot the slides without letting them dry and then apply 3% human serum (health hazard!) or pig serum a s a blocking agent.$ O- Q8 }, E9 e/ z y
8- incubate with the blocking for 10 min. ! z# @5 i6 F! h- a9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr. 7 R1 ?; d7 Q! a3 y4 L: x10- Wash twice. 5 @$ w+ T7 Z( J! t* R$ K( r9 m6 l( D0 ~
11- apply a mixture of secondary antibodies (see example below) for 45 min at RT. " q7 C" J( p$ h
12- Wash twice. ( ]5 P6 X5 C! z; U9 r: r13- add the mixture of primary antibodies and incubate for 15 min. 0 y$ ?* S/ L+ K5 ]1 E
14- Wash twice . * ?3 y0 Q$ y1 p; ~15- add the mixture of secondary antibodies and incubate for 15 min. / [3 l/ s; R7 L& {7 E3 `) V
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proceed with steps 16 through 19 if one of the secondary is biotin-conjugated. Else go to 19./ Q* S" f9 D# c% H
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16- dilute the avidin-fluorochrome in TBS/Tween and centrifuge for 15 min. 9 a- ^+ V* d. n9 G( M17- Wash thrice. 5 b. \9 ^2 r8 v1 j5 f' B( S
18- Incubate with the avidin for 15 min. 1 R O+ `) v) C% ^19- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount.( Z1 p2 \; f$ h( v
. B3 M3 O& B j8 u" d# UExample: Pirmary antibodies: rabbit anti-BCL-6 0.5µg/ml in PBS/BSA/Azide + mouse anti HA supn 1:100. 6 i8 G2 p! H$ R- uSecondary antibodies: biotin-conjugated goat anti rabbit (1:200) + TRITC conjugated goat anti-mouse (1:200). Alternatively, biotin-conjugated goat anti mouse (1:200) + goat anti rabbit TRITC (1:200) 2 a$ F6 Z) W) mAvidin FITC: 1:300 5 ?% A% q4 P8 P/ j) h3 F* GTo see the examples click on the thumbnail. $ {5 m; F, A1 D6 V/ d 3 Q5 {, B o) b( M8 T . k6 _) K8 {9 o